1. Field of the Invention
The present invention is directed to oligonucleotides which are capable of binding by H-bonding to a target sequence of nucleotides in the double stranded DNA of an invading organism, such as a virus, fungus, parasite, bacterium or tumor cell, and which carry at least two covalently linked electrophilic groups which after hybridization covalently bind to the target sequence of nucleotides, with the result that replication and/or expression of the target sequence is prevented. Alternatively, the oligonucleotides of the present invention can bind in vitro by H-bonding to a target sequence of duplex DNA which is to be cleaved at the target location . Alkylation of both strands of the duplex DNA enhances its susceptibility to cleavage at the alkylation sites and therefore provides a tool for DNA mapping or similar investigative or analytical purposes.
2. Description of the Prior Art
Oligodeoxynucleotides (ODNs) have great potential as sequence specific pharmaceutical agents for the inhibition of gene expression. Chemically synthesized ODNs may inhibit the expression of specific gene products through formation of duplexes upon hybridization with complementary messenger RNAs (mRNAs). More specifically, these "antisense" ODNs are believed to inhibit the processing or translation of message primarily through an RNase H-mediated cleavage of the target mRNA sequence. Because of this inhibitory effect, antisense ODNs may be useful as anti-viral, anti-parasitic, and anti-cancer agents. However, "antisense" technology is beset with certain fundamental disadvantages relating, for example, to degradation of antisense ODNs by nuclease enzymes, and uptake (or lack of uptake) by cells. To improve their properties, modified antisense ODNs, such as ODNs with modified backbones (oligonucleoside methylphosphonates and phosphorothioates) have been prepared. It has been found however, that improvement in some properties, such as resistance to nuclease enzymes frequently has deleterious effects on other properties, such as cellular uptake and loss of specificity.
Another approach to improve the effectiveness of antisense ODNs involves covalently attaching moieties to the antisense ODNs which moieties interact directly with the target RNA upon hybridization and therefore potentiate the antisense activity of the ODN. Groups employed in this regard are intercalating groups, and groups which covalently link with the target RNA after hybridization.
Anti-gene ODNs
A variation of the "antisense" approach to rational drug design is termed "anti-gene". Whereas antisense ODNs target single stranded MRNA, anti-gene ODNs hybridize with and are capable of inhibiting the function of double-stranded DNA. More specifically, anti-gene ODNs form sequence-specific triple-stranded complexes with a double stranded DNA target and thus interfere with the replication or transcription of selected target genes. As is known, except for certain RNA viruses and nucleic acid-free viroids, DNA is the repository for all genetic information, including regulatory control sequences and non-expressed genes, such as dormant proviral DNA genomes. In contrast, the target for antisense ODNs, MRNA, represents a very small subset of the information encoded in DNA. Thus, anti-gene ODNs have broader applicability and are potentially more powerful than antisense ODNs that merely inhibit mRNA processing and translation.
Anti-gene ODNs in the nuclei of living cells can form sequence-specific complexes with chromosomal DNA. The resultant triplexes have been shown to inhibit restriction and/or transcription of the target double stranded DNA. Based on the known stabilities of the two target nucleic acid species (i.e., DNA and RNA), anti-gene interference with DNA functioning has longer lasting effects than the corresponding antisense inhibition of mRNA function.
Anti-gene therapy may be based on the observation that under certain conditions DNA can form triple-stranded complexes. In these triple-standed complexes, the third strand resides in the major groove of the Watson-Crick base paired double helix, where it hydrogen bonds to one of the two parental strands. A binding code governs the recognition of base pairs by a third base (see allowed triplets below, Hoogsteen pairing). In each case, the third strand base is presented first and is followed by the base pair in the Watson-Crick duplex.
allowed triplets: A-A-T G-G-C T-A-T C-G-C
Certain limitations of this base pair recognition code are apparent from the allowed triplets. First, there is no capability for the recognition of T-A and C-G base pairs; hence, triple strand formation is restricted to runs of homopurine bases on one strand and homopyrimidine bases on the other strand of the duplex. In other words, the third strand or ODN binds only to one strand of the duplex and can only bind to purines. Second, if cytosine is in the third strand ("C"), it must be protonated to be able to hydrogen bond to the guanine of a G-C base pair. The pKa for protonation of cytosine is 4.6, suggesting that at physiological pH the stability of C-G-C triads is likely to be impaired. Third, in all cases triads are maintained by two hydrogen bonds between the third strand base and the purine residue of the duplex base pair. Hence, triple-stranded complexes are generally less stable than the parental double-stranded DNA, which is maintained by a combination of two (A-T) or three (G-C) hydrogen bonds between purine and pyrimidine pairs. (Watson-Crick motif).
An important disadvantage of triple strand formation as discussed above is the relatively slow kinetics of triple strand formation. However, triple strand formation can be catalyzed in cells by recombinase enzymes which are practically ubiquitous in cells and whose existence is well known in the art. In addition to a much faster rate of triple strand formation, recombinase enzyme-catalyzed triple strand formation also provides the advantage of universal sequence recognition (in contrast to the A-T and G-C recognition limitation associated with non-enzyme-mediated triple strand formation). More specifically, the recombinase enzyme-mediated recognition motif recognizes all four base pairs, thereby allowing targeting of any double stranded DNA sequence. Second, the nucleoprotein filament, which is the complex formed between a recombinase enzyme and the single-stranded ODN, searches for target double strand DNA homology much more efficiently than does a small naked anti-gene ODN, thus decreasing the concentration of anti-gene ODN required for efficient triple strand complex formation. Third, due to the hydrogen bonding patterns and the novel helical twist involved in enzyme-mediated recognition, the resultant triple strand complex is stable at physiological pH. Fourth, since the cellular recombinational pathway is being harnessed, the DNA in higher order chromatin structures will be accessible for targeting.
The ability to conduct an efficient homology search is a significant advantage. Preliminary data (F. M. Orson et al., Nucl. Acids Res. 19:3435-41, 1991) indicate that ODNs are inefficient at scanning double stranded DNA for complementary homopurine sequences. In contrast, a classical hybridization between two complementary single strands would occur within seconds, rather than hours. Since the human genome contains over 3.times.10.sup.9 base pairs, the homology search time may be inordinately long, especially if anti-gene ODNs are used at relatively low concentration. The use of presynaptic nucleoprotein filaments, such as those formed between single stranded DNA and recA, that bind weakly to and move rapidly along double stranded DNA effectively reduces the homology search from a three dimensional to a two dimensional process. Furthermore, upon homologous registry with the double strand, the nucleoprotein filament will more likely produce a triple strand complex than the corresponding interaction of double strand and a naked single strand.
Because of these factors, triple strand formation between a recA-coated, single stranded ODN and an homologous double strand occurs at a reaction rate that exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation of complementary single strands under standard hybridization conditions.
A first demonstration of the concept of using sequence-specific, antisense oligonucleotides as regulators of gene expression and as chemotherapeutic agents was described by Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA, 75:280 (1978). These authors showed that a small antisense oligodeoxynucleotide probe can inhibit replication of Rous Sarcoma Virus in cell culture, and that RSV viral RNA translation is inhibited under these conditions (Stephenson et al., Proc. Natl. Acad. Sci. USA 75:285 (1978)). Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83:4143 (1986), have also shown that oligonucleotides complementary to portions of the HIV genome are capable of inhibiting protein expression and virus replication in cell culture. Inhibition of up to 95% was obtained with oligonucleotide concentrations of about 70 .mu.M. Importantly, they showed with labeled phosphate studies that the oligonucleotides enter cells intact and are reasonably stable to metabolism.
The concept of covalently linking an inhibitor molecule to a target (such as binding an ODN to an target sequence with a cross-linking arm,) is related to the pioneering work of B. R. Baker, "Design of Active-Site-Directed Irreversible Enzyme Inhibitors," Wiley, New York, (1967), who used what was termed "active-site-directed enzyme inhibitors" in chemotherapeutic applications. The concept of incorporating a crosslink in an oligonucleotide has been sporaidically discussed by several authors. For example, Knorre and Vlassov, Prog. Nucl. Acid Res. Mol. Biol., 32:291 (1985), have discussed sequence-directed crosslinking ("complementary addressed modification") using an N-(2-chloroethyl)-N-methylaniline group attached to either the 3'- or 5'-terminus of oligonucleotides. Summerton and Bartlett, J. Mol. Biol., 122:145 (1978) have shown that an 8-atom chain, attached to a cytosine residue at its C-4 position and terminating in the highly reactive bromomethyl ketone group, can crosslink to the N-7 of guanosine. Webb and Matteucci, Nucleic Acids Res., 14:7661 (1986), have prepared oligonucleotides containing a 5-methyl-N,N-ethanocytosine base which is capable of slow crosslinking with a complementary strand. In a conceptually related alkylation via a linker arm within a DNA hybrid, Iverson and Dervan, Proc. Natl. Acad. Sci. USA, 85:4615 (1988), have shown opposite strand methylation, triggered by BrCN activation of a methylthio ether, predominately on a guanine base located two pairs from the base bearing the linker. Vlassov et al. in Gene 72 (1988) 313-322, describe sequence specific binding and alkylation of plasmid DNA with oligodeoxynucleotide derivatives containing 2-chloroethyl-N-methyl amino phenyl residues. Similar cross-linking, using different cross-linking agent was described by Shaw et al., J. Am.Chem. Soc. 1991, 113, 7765-7766.
Further information pertaining to ODNs, chemically modified ODNs and their ability to affect or inhibit replication or translation of a target sequence of DNA or RNA can be found in European Patent Application No. 86309090.8, PCT publication WO8707611, U.S. Pat. No. 4,599,303, EP 0259186, PCT publication WO8503075, German Patent DE3310337, and in the publications Blake et al., Biochemistry 24:6139 (1985); Umlauf et al., "Triple-helical DNA Pairing Intermediates Formed by recA Protein,", Biol. Chem., 265(28), 16898-16912 (1990); and Thuong et al., "Chemical synthesis of natural and modified oligodeoxynucleotides.", Biochimie, 1985, 67, 673-684.
DNA Mapping
In addition to chemotherapy or potential chemotherapy utilizing ODNs or modified ODNS, a broad field has developed in the prior art for DNA mapping (gene mapping), that is, for in vitro determination of DNA sequence or partial DNA sequence. An important step in such DNA sequencing (gene mapping) is the cleavage of the target DNA into smaller fragments. The modified ODNs of the present invention also have utility in this field.